Flavin-dependent azoreductases have been applied to a wide range of tasks from decolorizing numerous azo dyes to releasing azo-conjugated prodrugs. A general narrative reiterated in much of the literature suggests that this enzyme promotes sequential reduction of both the azo-containing substrate and its corresponding hydrazo product to release the aryl amine components while consuming two equivalents of NAD(P)H. Indeed, such aryl amines can be formed by incubation of certain azo compounds with azoreductases, but the nature of the substrates capable of this apparent azo bond lysis remained unknown. We have now prepared a set of azobenzene derivatives and characterized their turnover and products after treatment with azoreductase from Escherichia coli to discover the structural basis regulating aryl amine formation. Without resonance donation by aryl substituents, reduction ceases at the hydrazo product. This indicates that azoreductases do not act on the hydrazo bond. Instead, aryl amine formation depends on a spontaneous hydrazo bond lysis that is promoted by resonance stabilization and subsequent reduction of the quinone-like intermediate by azoreductase. Experimental and computational approaches confirm the substituent dependence of this process. With knowledge of this requirement, full release of aryl amines from azo-conjugates can now be designed and applied with confidence.

Yu-Ju Peng

Bing Xu

Steven E. Rokita

ACS Chemical Biology

ACS 化学生物学为广泛涵盖化学和生物学之间接口的研究提供了一个快速交流的国际论坛。该期刊还作为一个论坛，促进生物学家和化学家之间的交流，从而转化为新的研究机会和发现。将发表结果，其中分子推理已被用于通过体外研究、细胞生物学方法或有机体研究来探索问题。我们欢迎对蛋白质、核酸、糖、脂质和非生物聚合物的机械研究。该杂志服务于一个大型科学界，从化学和生物学角度探索细胞功能。据了解，提交的作品是基于原始结果，之前没有发表过。

ACS Chemical Biology provides an international forum for rapid communication of research that broadly encompasses the interface between chemistry and biology. The journal serves as a platform to facilitate interactions between biologists and chemists, leading to new research opportunities and discoveries. We welcome the publication of results where molecular reasoning has been applied to explore problems through in vitro studies, cell biology methods, or organismal research. We particularly encourage mechanistic studies on proteins, nucleic acids, carbohydrates, lipids, and non-biological polymers. This journal serves a large scientific community that explores cellular functions from both chemical and biological perspectives. Please note that submitted works must be based on original results and have not been previously published.

ACS Chemical Biology 是一个国际论坛，广泛涵盖化学与生物学交叉领域的研究。该期刊旨在促进生物学家与化学家之间的交流，从而催生新的研究机会和发现。我们欢迎发表利用分子推理，通过体外研究、细胞生物学方法或有机体研究探索问题的研究成果。我们特别关注对蛋白质、核酸、糖、脂质及非生物聚合物的机制研究。该期刊服务于一个庞大的科学社区，致力于从化学和生物学的角度探索细胞功能。请注意，提交的作品必须基于原创结果，且未曾发表过。

Category	Quartile
生物学	2
生物学, 生化与分子生物学	3

Breaking the Myth of Enzymatic Azoreduction

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Journal Info

Category	Quartile
BIOCHEMISTRY & MOLECULAR BIOLOGY	2